Dear Editor,

Acute myeloid leukemia (AML) is frequently associated with FLT3 mutations, which are present in approximately 30% of cases. These mutations are associated with a poor prognosis, frequent relapses, and reduced overall survival [1, 2]. FLT3 mutations lead to constitutive activation of the tyrosine kinase and subsequent downstream proliferation cascades, such as those regulated by STAT5, resulting in uncontrolled proliferation of leukemic cells [3, 4]. Small molecule tyrosine kinase inhibitors (TKIs) that specifically target FLT3 have improved survival rates in patients with FLT3-mutated AML [5]. Although these agents have demonstrated significantly better survival outcomes compared to patients treated with placebos or salvage chemotherapy, a substantial proportion of patients still experience disease relapse, primarily due to the development of primary and secondary resistance to FLT3-TKIs [6,7,8,9].

Cyclin-dependent kinase (CDK) inhibitors have shown therapeutic effects by targeting the disruption in cell cycles in cancer cells. Several CDK7 inhibitors have demonstrated clinical benefits in the treatment of advanced solid tumors and CDK7 is considered an ideal candidate for inhibiting MYC amplification and regulating mitochondrial proteins [10, 11]. The majority of CDK7 inhibitors are associated with alterations in the phosphorylation of RNA polymerase II [12]. We have reported that YPN-005, a highly selective CDK7 inhibitor, induces apoptotic cell death and reduces RNA polymerase II phosphorylation, rather than causing cell cycle arrest in the G1/S or G2/M phases, in AML cell lines [13]. Notably, significant inhibition of cell growth was observed in both AML cell lines and patient-derived primary AML cells harboring FLT3 mutations. This inhibition was accompanied by downregulation of FLT3 and STAT5 signaling at both the protein and mRNA levels. We found that YPN-005 exhibited superior apoptotic efficacy in FLT3 internal tandem duplication (ITD)-mutated AML cell lines, orthotopic mouse models, and mononuclear cells derived from the bone marrow of patients. Therefore, in this study, we investigated whether a synergistic effect could be achieved through combination therapy with an FLT3 inhibitor.

We first examined whether YPN-005 exerted synergistic antileukemic effects with FLT3-TKIs in AML cell lines harboring FLT3-ITD mutation. FLT3-ITD-mutated AML cells were treated with YPN-005 and three FDA-approved FLT3-TKIs—midostaurin, quizartinib, and gilteritinib—either as single agents or in combination. To evaluate the significance of the synergistic activities between YPN-005 and the FLT3-TKIs, we performed pairwise drug combination assays in the FLT3-ITD-mutated cell lines. The optimal concentration for confirming combination therapy was determined as the concentration that induced 30–70% growth inhibition for each drug. Cell viability assay was performed after 72 hours of treatment, and the resulting survival rates were analyzed using the Bliss independence model to assess synergism (Supplementary Table 1). All FLT3-TKIs demonstrated synergistic inhibition of cell proliferation when combined with YPN-005 in four FLT3-ITD-mutated cell lines: Ba/F3-ITD, MV4-11, MOLM-13, and MOLM-14, with Bliss indices > 10 (Fig. 1a). Furthermore, the synergistic effect was observed even when each drug was administered in a time-dependent manner (Supplementary Fig. 1).

Fig. 1: Synergistic effects of combination treatments of YPN-005 and FLT3-TKIs on FLT3-ITD-mutated AML cells.
figure 1

The combination matrix shows the interactions between YPN-005 and the corresponding FLT3-TKI. Synergy scores, calculated in accordance with the Bliss independence model, are shown in the combination matrix. The largest numeral in each box represents the synergy score (Blue color); negative values indicate antagonism (Red color). Bliss indices > 10 indicate synergism (a). Induction of apoptosis by the combined treatment of FLT3-ITD-mutated AML cell lines with YPN-005 and FLT3-TKIs. Apoptosis was evaluated using Annexin V/PI staining after combination treatment with YPN-005 and FLT3-TKIs. Flow cytometry analysis was performed after incubation with each agent for 72 h. *p < 0.05, **p < 0.01, ***p < 0.001 (b). The protein expression of cleaved PARP and cleaved caspase-3, -8, and -9 was determined by immunoblotting, combination treatment with YPN-005 and quizartinib after 6 h. The arrows indicate the bands formed by each of the subunits of the corresponding cleaved protein (c). In vivo evaluation of the synergistic effects of YPN-005 and FLT3-TKIs. NSG mice were engrafted with MOLM-13 cells expressing GFP and either treated with the vehicle, 2 mg/kg YPN-005, 2 mg/kg quizartinib, 30 mg/kg gilteritinib, or both YPN-005 and quizartinib or gilteritinib (n = 5 per group). The vehicle (saline) group was compared against each treatment group. The in vivo imaging system was used to evaluate the effects of each treatment in the orthotopic xenograft mouse model (d). Survival analysis was estimated using the Kaplan–Meier method. Kaplan–Meier curves with log rank (Mantel-Cox) tests were obtained for survival determination. **p < 0.01 was considered statistically significant (e). Inhibition of the colony-forming ability of primary AML with FLT3-ITD cells by combined treatment with YPN-005 and quizartinib compared to that after single agent treatments. The images are representative micrographs of the resulting colony forming units from control and treated AML cells from three different patients (f).

Full size image

To determine whether the combinational efficacy of coadministering YPN-005 and FLT3-TKIs was related to apoptosis induction, we assessed the apoptotic cell population and cell cycle using FACS and evaluated the activities of caspase and PARP through immunoblot analysis. Flow cytometry analysis revealed that the combined treatment of YPN-005 and FLT3-TKIs significantly induced apoptotic cell death compared to the effects observed in cells treated with single agents (Fig. 1b). However, cell cycle arrest, which is one of the mechanisms mediated by CDK7, was not significantly induced (Supplementary Fig. 2). Additionally, immunoblotting analysis showed the induction of PARP and the cleavages of caspase-9, -8, and -3 after a 6-hour combined treatment with YPN-005 and FLT3-TKIs (Fig. 1c). To validate the in vivo efficacy of the combination treatment, we established a leukemic orthotopic mouse model using GFP-expressing MOLM-13 cells with FLT3-ITD mutations. After administering YPN-005 in combination with quizartinib and gilteritinib, either alone or in combination, we assessed bioluminescence imaging and survival rates. The combination treatment in orthotopic mice carrying FLT3-ITD-mutated AML cells effectively reduced the leukemic burden (Fig. 1d), and overall survival significantly improved in cotreated mice compared to those receiving single-agent treatments (Fig. 1e). Bone marrow (BM) mononuclear cells harvested from three patients with FLT3-ITD-mutated AML were evaluated for efficacy using the methylcellulose CFU assay (Supplementary Table 2). The coadministration of YPN-005 and FLT3-TKIs led to a reduced colony-forming capacity of primary AML cells (Fig. 1f). These data demonstrate that YPN-005 and FLT3-TKIs synergistically induce growth inhibition in AML cells with FLT3-ITD mutations in vitro, in vivo, and ex vivo.

We then aimed to identify the common mechanisms underlying the synergistic effects between YPN-005 and FLT3-TKIs. By analyzing the expression profiles of each drug’s targets within the combination, we evaluated the potential for establishing a mechanistically complementary relationship. As anticipated, levels of phosphorylated FLT3 and STAT5 decreased following quizartinib treatment, while RNA polymerase II expression was reduced after YPN-005 treatment (Fig. 2a). Notably, the combined treatment of YPN-005 and quizartinib resulted in a significant reduction in both phosphorylated FLT3 and RNA polymerase II levels compared to those observed with single-agent treatments.

Fig. 2: c-MYC is downregulated by YPN-005 and FLT3-TKI combination treatments.
figure 2

Decreased expression of FLT3 signaling molecules and RNA polymerase by the combined treatment of FLT3-ITD-mutated AML cell lines with YPN-005 and FLT3-TKIs. Immunoblot analysis of the expression of FLT3 signaling pathway proteins and RNA polymerase in MV4-11, MOLM-13, and MOLM-14 cells treated with 5 nM quizaritinib, 20 nM YPN-005, or a combination of both for 6 h (a). Induction of mitochondrial damage by combination treatment with YPN-005 and FLT3-TKIs. Electron micrographs of MV4-11 cells either (a) untreated or treated with (b) 5 nM quizartinib (c) 20 nM YPN-005, or (d) a combination of both agents for 72 h. Scale bars = 0.2 µm (b). Changes in mitochondrial membrane potential (MMP, ΔΨm) in MV4-11 cells stained with tetramethylrhodamine ethyl ester were determined by flow cytometry. Cells treated with 50 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were used as positive controls. Depolarization of the MMP was observed in cells treated with 10 nM YPN-005, 20 nM midostaurin, 1 nM quizartinib, 5 nM gilteritinib, or a combination of YPN-005 and an FLT3-TKI for 72 h. MOCK, negative control cells treated with Dimethyl Sulfoxide (DMSO) only (c). The bar graph is illustrating the percent of MMP depolarization in MV4-11 cells treated YPN-005, FLT3-TKIs or both agents as compare to control group. *p < 0.05, ***p < 0.001 (d). Protein expression in FLT3-ITD-mutated AML cells was evaluated using immunoblotting after combination treatment with 10 nM YPN-005 and 1 nM quizartinib (MV4-11 and MOLM-13 cell lines), or 12 nM YPN-005 and 2 nM quizartinib (MOLM-14 cell line) for 6 h (e). Immunoblot detection of c-MYC protein in mouse bone marrow mononuclear cells after a 21 d treatment with either 2 mg/kg YPN-005, 2 mg/kg quizartinib, 30 mg/kg gilteritinib, or a combination of YPN-005 and an FLT3-TKI (n = 3 per group) (f). Immunoblot band density was measured using CSAnalyzer4 and normalized to that of β-actin (g).

Full size image

Previous studies have reported that both FLT3-ITD and CDK7 are directly involved in the mechanisms that regulate mitochondrial function [14, 15]. To assess the impact of YPN-005 and FLT3-TKIs on structural changes in mitochondria, we examined mitochondrial morphology and integrity in MV4-11 cells using electron microscopy (EM) after a 24-hour exposure to 20 nM YPN-005 and 5 nM quizartinib. Our EM analysis showed notable mitochondrial abnormalities, including swollen mitochondria and disorganized cristae, following the combined treatment (Fig. 2b). While similar effects were observed in MV4-11 cells treated with single agents, the combination treatment resulted in a more severe disruption of mitochondrial structure. Since mitochondrial membrane potential (MMP) reflects the functional maintenance of mitochondria, we monitored MMP levels in FLT3-ITD-mutated AML cells treated with YPN-005 and FLT3-TKIs. While YPN-005 and FLT3-TKI single-agent treatments induced a 20 to 40% reduction in MMP in MV4-11 cells, combined treatments with YPN-005 and FLT3-TKIs led to an additional 20 to 30% decrease in MMP (Fig. 2c, d). Similar results were observed in Ba/F3-ITD, MOLM-13, and MOLM-14 cells (Supplementary Fig. 3a–f).

To investigate the molecular mechanisms by which the combined administration of YPN-005 and FLT3-TKIs induces apoptosis through mitochondrial dysfunction in FLT3-ITD-mutated AML cells, we conducted mRNA sequencing before and after cell exposure to the drugs. Following the cotreatment of MV4-11 cells with YPN-005 and quizartinib, we selected 100 upregulated and 100 downregulated genes based on their log2 fold change among a total of 26,255 genes, in relation to their expression in cells treated with the vehicle alone (Supplementary Fig. 4). Gene set enrichment analysis (GSEA) indicated that the combination of YPN-005 and quizartinib altered the expression of mitochondrial-related pathway genes compared to the control in MV4-11 cells (Supplementary Fig. 5). Subsequently, we generated a protein-protein interaction network using Cytoscape and data from the STRING database, incorporating all differentially expressed genes. This network comprised 142 genes with 200 interactions, featuring MYC as the central hub (Supplementary Fig. 6). The combination therapy resulted in a significant reduction in c-MYC gene expression compared to that observed after YPN-005 or FLT3-TKI monotherapy in FLT3-ITD-mutated AML cell lines (Fig. 2e, Supplementary Fig. 7). To confirm the decrease in c-MYC expression, mononuclear cells were isolated from the BM of the GFP-expressing FLT3-ITD-mutated MOLM-13 orthotopic mouse model. Protein was extracted from these cells, and the reduction in c-MYC expression was confirmed to be significant (Fig. 2f, g). These findings indicate that the combination of YPN-005 and FLT3-TKI treatment effectively reduces c-MYC expression associated with mitochondrial dysfunction in FLT3-ITD-mutated AML cells.

In conclusion, our study demonstrates that a combined treatment approach involving the CDK7 inhibitor YPN-005 and FLT3-TKIs exhibits synergistic antileukemic effects against FLT3-ITD-mutated AML cells by inducing apoptotic cell death. The observed apoptotic effects were associated with mitochondrial dysfunction and a reduction in c-MYC expression. These findings suggest that targeting both CDK7 and FLT3 may represent a promising therapeutic strategy to overcome drug resistance and enhance clinical outcomes in patients with FLT3-ITD-mutated AML.